Volatile matrices for matrix-assisted laser desorption/ionization mass spectrometry

ABSTRACT

A sample preparation method is disclosed for volatilization and mass spectrometric analysis of nonvolatile high molecular weight molecules. Photoabsorbing molecules having significant sublimation rates at room temperature under vacuum, and preferably containing hydroxy functionalities, are disclosed for use as matrices in matrix-assisted laser desorption/ionization mass spectrometry. The samples are typically cooled in the mass spectrometer to temperatures significantly below room temperature.

This application is a continuation of and claims the benefit of U.S.application Ser. No. 09/086,993 filed May 29, 1998, now U.S. Pat. No.6,104,028.

The government may own rights in the present invention pursuant to GrantNo. R01 HG-00174 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to volatile photoabsorbing matrices having a lowsublimation temperature for use in the mass spectrometric analysis oflarge, nonvolatile molecules. This invention also relates to methods forpreparing samples containing large, nonvolatile analyte molecules forlaser desorption mass spectrometry employing such matrices.

2. Description of Related Art

Approximately 4,000 human disorders are attributed to genetic causes.Hundreds of genes responsible for various disorders have been mapped,and sequence information is being accumulated rapidly. A principal goalof the Human Genome Project is to find all genes associated with eachdisorder. The definitive diagnostic test for any specific geneticdisease (or predisposition to disease) will be the identification ofpolymorphic variations in the DNA sequence of affected cells that resultin alterations of gene function. Furthermore, response to specificmedications may depend on the presence of polymorphisms. Developing DNA(or RNA) screening as a practical tool for medical diagnostics requiresa method that is inexpensive, accurate, expeditious, and robust.

Genetic polymorphisms and mutations can manifest themselves in severalforms, such as point polymorphisms or point mutations where a singlebase is changed to one of the three other bases; deletions where one ormore bases are removed from a nucleic acid sequence and the basesflanking the deleted sequence are directly linked to each other;insertions where new bases are inserted at a particular point in anucleic acid sequence adding additional length to the overall sequence;and expansions and reductions of repeating sequence motifs. Largeinsertions and deletions, often the result of chromosomal recombinationand rearrangement events, can lead to partial or complete loss of theactivity of a gene. Of these forms of polymorphism, in general the mostdifficult type of change to screen for and detect is the pointpolymorphism because it represents the smallest degree of molecularchange.

Although a number of genetic defects can be linked to a specific singlepoint mutation within a gene, e.g. sickle cell anemia, many are causedby a wide spectrum of different mutations throughout the gene. A typicalgene that might be screened could be anywhere from 1,000 to 100,000bases in length, though smaller and larger genes do exist. Of thatamount of DNA, only a fraction of the base pairs actually encode theprotein. These discontinuous protein coding regions are called exons andthe remainder of the gene is referred to as introns. Of these two typesof regions, exons often contain the most important sequences to bescreened. Several complex procedures have been developed for scanninggenes in order to detect polymorphisms. These procedures are applicableto both exons and introns.

In terms of current use, most of the methods to scan or screen genesemploy slab or capillary gel electrophoresis for the separation anddetection step in the assays. Gel electrophoresis of nucleic acidsprimarily provides relative size information based on mobility throughthe gel matrix. If calibration standards are employed, gelelectrophoresis can be used to measure absolute and relative molecularweights of large biomolecules with some moderate degree of accuracy;even then, the accuracy is typically only 5% to 10%. Also the molecularweight resolution is limited. In cases where two DNA fragments with theidentical number of base pairs can be separated, for example, by usinghigh concentration polyacrylamide gels, it is still not possible toidentify which band on a gel corresponds to which DNA fragment withoutperforming secondary labeling experiments. Thus, gel electrophoresistechniques can only determine size and cannot provide any informationabout changes in base composition or sequence without performing morecomplex sequencing reactions. Gel-based techniques, for the most part,are dependent on labeling or staining methods to visualize anddiscriminate between different nucleic acid fragments.

Many methods in use today capable of screening broadly for geneticpolymorphisms suffer from technical complication and are labor and timeintensive. Single strand conformational polymorphism (SSCP) (Orita etal., 1989), denaturing gradient gel electrophoresis (DGGE) (Abrams etal., 1990), chemical cleavage at mismatch (CCM) (Saleeba and Cotton,1993), enzymatic mismatch cleavage (EMC) (Youil et al., 1995), andcleavage fragment length polymorphism (CFLP) procedures are currentlygel-based, making them cumbersome to automate and perform efficiently.Thus, there is a need for new methods that can provide cost effectiveand expeditious means for screening genetic material in an effort todetect genetic mutations and diagnose related medical conditions simply,quickly, accurately, and inexpensively.

Another approach that is having some success is to employ massspectrometry to screen for and detect genetic mutations as well as tosequence nucleic acids. In order to measure the mass of nonvolatile highmolecular weight molecules, typically greater than 1000 Da, in a massspectrometer, the analyte molecules must first be volatilized orconverted into gas-phase ions. Although direct laser desorption of theneat analyte is one approach to volatilizing the molecule, the energydeposited into the analyte may induce fragmentation and lead to resultsthat are ambiguous or difficult to analyze. The late 1980's saw the riseof two new mass spectrometric techniques which are potentially suitablefor genetic screening tests by successfully measuring the masses ofintact very large biomolecules, namely, matrix-assisted laserdesorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS)(Tanaka et al., 1988; Spengler et al., 1989) and electrospray ionization(ES) combined with a variety of mass analyzers. The MALDI massspectrometric technique can also be used with methods other thantime-of-flight, for example, magnetic sector, Fourier-transform ioncyclotron resonance, quadrupole, and quadrupole trap.

MALDI-TOF MS involves laser pulses focused on a small sample plate onwhich analyte molecules (i.e. nucleic acids) are embedded in either asolid or liquid matrix which is typically a small, highly absorbingmaterial, such as a small aromatic organic molecule. The volatilizationof intact fragile molecules benefits from the use of matrix-assistedlaser desorption ionization because the radiative energy from the laserpulse is coupled indirectly into the analyte through the matrixmolecules. Typically, the analyte molecules are crystallized with alarge molar excess of a photoabsorbing matrix (see U.S. Pat. Nos.4,920,264 and 5,118,937, incorporated herein by reference). An advancein MALDI analysis of polynucleotides was the discovery of3-hydroxypicolinic acid (3-HPA) as a suitable matrix for mixed-baseoligonucleotides (Wu, et al., 1993).

The laser pulses transfer energy to the matrix causing a microscopicablation and concomitant ionization of the analyte molecules, producinga gaseous plume of intact, charged nucleic acids in single-strandedform. It is thought that upon laser excitation the matrix molecules arerapidly heated and ejected into the gas phase, carrying analytemolecules into the expansion plume of molecules and ions. It is thoughtthat gas-phase ion-molecule collisions subsequently ionize the neutralanalyte molecules in the near-surface region, often via proton transfer.The matrix thus functions as both an energy- and charge-transferspecies. If double-stranded nucleic acids are analyzed, the MALDI-TOF MStypically results in detection of mostly charged denaturedsingle-stranded nucleic acids.

The ions generated by the laser pulses are accelerated to a fixedkinetic energy by a strong electric field and then passed through anelectric field-free region in vacuum, traveling with a velocitycorresponding to their respective mass-to-charge ratios (m/z). Thus, thesmaller m/z ions will travel through the vacuum region faster than thelarger m/z ions thereby causing a separation. At the end of the electricfield-free region, the ions collide with a detector that generates asignal as each set of ions of a particular mass-to-charge ratio strikesthe detector. Usually for a given assay, 10 to 100 mass spectraresulting from individual laser pulses are summed together to make asingle composite mass spectrum with an improved signal-to-noise ratio.

The mass of an ion (such as a charged nucleic acid) is measured by usingits velocity to determine the mass-to-charge ratio by time-of-flightanalysis. In other words, the mass of the molecule directly correlateswith the time it takes to travel from the sample plate to the detector.The entire process takes only microseconds. In an automated apparatus,tens to hundreds of samples can be analyzed per minute. In addition tospeed, MALDI-TOF MS has one of the largest mass ranges for massspectrometric devices. The current mass range for MALDI-TOF MS is from 1to 1,000,000 Da (measured recently for a protein) (Nelson et al., 1995).

The performance of a mass spectrometer is measured by its sensitivity,mass resolution and mass accuracy. Sensitivity is measured by the amountof material needed; it is generally desirable and possible with massspectrometry to work with sample amounts in the femtomole and lowpicomole range. Mass resolution, m/Δm, is the measure of an instrument'sability to produce separate signals from ions of similar mass. Massresolution is defined as the mass, m, of an ion signal divided by thefull width of the signal, Δm, usually measured between points ofhalf-maximum intensity. Mass accuracy is the measure of error indesignating a mass to an ion signal. The mass accuracy is defined as theratio of the mass assignment error divided by the mass of the ion andcan be represented as a percentage.

To be able to detect any point polymorphism directly by MALDI-TOF massspectrometry, one would need to resolve and accurately measure themasses of nucleic acids in which a single base change has occurred (incomparison to the wild type nucleic acid). A single base change can be amass difference of as little as 9 Da. This value represents thedifference between the two bases with the closest mass values, A and T(A=2′-deoxyadenosine-5′-phosphate=313.19 Da;T=2′-deoxythymidine-5′-phosphate=304.20 Da;G=2′-deoxyguanosine-5′-phosphate=329.21 Da; andC=2′-deoxycytidine-5′-phosphate=289.19 Da). If during the mutationprocess, a single A changes to T or a single T to A, the mutant nucleicacid containing the base transversion will either decrease or increaseby 9 Da in total mass as compared to the wild type nucleic acid. Formass spectrometry to directly detect these transversions, it musttherefore be able to detect a minimum mass change, Δm, of approximately9 Da.

For example, in order to fully resolve (which may not be necessary) apoint-mutated (A to T or T to A) heterozygote 50-base single-strandedDNA fragment having a mass, m, of ˜15,000 Da from its corresponding wildtype nucleic acid, the required mass resolution is m/Δm=15,000/9≈1,700.However, the mass accuracy needs to be significantly better than 9 Da toincrease quality assurance and to prevent ambiguities where the measuredmass value is near the half-way point between the two theoreticalmasses. For an analyte of 15,000 Da, in practice the mass accuracy needsto be Δm˜±3 Da=6 Da. In this case, the absolute mass accuracy requiredis (6/15,000)*100=0.04%. Often a distinguishing level of mass accuracyrelative to another known peak in the spectrum is sufficient to resolveambiguities. For example, if there is a known mass peak 1000 Da from themass peak in question, the relative position of the unknown to the knownpeak may be known with greater accuracy than that provided by anabsolute, previous calibration of the mass spectrometer.

In addition, the ability to separate DNA fragments (1) differing in onlyone base in length and (2) of reasonable length (e.g., of sizescorresponding to at least primer size, around 20 to 30 bases or so up toabout 50 bases in length) is critical to achieving even rudimentary DNAsequencing by MALDI-MS. For laser desorption mass spectroscopytechniques to successfully analyze macromolecules requires that onestably laser-desorb molecules into a vapor phase, and separate anddetect (and thereby determine the mass of) the volatilized molecules bymass spectroscopy. The ability to stably desorb the macromoleculedepends on the availability of a suitable light absorbing matrix thatwill allow one to stably laser-desorb DNA molecules from a solid stateto a gaseous state, and permit separation of DNA molecules having only anucleotide or so difference in length. Putting that into perspective,the difference in mass between a polynucleotide having 30 versus 31nucleotide represents about a 3% difference in mass (about 9610 v. 310,assuming an average m.w. of 310 for each nucleotide). If one appliesthis to a DNA molecule of 100 nucleotides in length, a modest sequenceby DNA sequencing standards, the separation system must distinguishamong DNA molecules differing by only 1% in mass.

Thus, there is a need for the development of MS techniques and relatedmaterials for practicing these techniques that have enhanced resolution,accuracy, and sensitivity. The ability to stably desorb the moleculefrom a solid matrix that absorbs light at the laser wavelength, withoutradiation damage and fragmentation of the sample is particularlyimportant as fragmentation can lead to complex spectra and decreasedresolution and sensitivity.

Although MALDI generates less energetic analyte ions than direct laserdesorption, thus decreasing the thermal degradation of the analyte, theions nevertheless contain significant internal energy, which may resultin fragmentation. Among the few matrix molecules that have been found todesorb/ionize intact DNA, 3-HPA is currently the most widely used (Wu etal., 1993; Wu et al., 1994)). Using a matrix mixture of 3-HPA withpicolinic acid, oligonucleotides have been detected that are greaterthan 500 bases (up to about 200 kDa) in length (Tang et al., 1994; Liuet al., 1995). However, as the length of the oligonucleotide increases,the mass resolution is degraded by widening kinetic energy spreads,prompt fragmentation, delayed fragmentation (metastable decay), and theformation of matrix adducts. Thus, there is a need to develop MSmaterials and methods that minimize fragmentation of the analyte ionsduring the MALDI process, extend the accessible mass range for massspectrometric detection, and enhance the utility of the MS techniques.

SUMMARY OF THE INVENTION

It is therefore a goal of the present invention to provide compositionsand methods relating to the preparation of samples containingnonvolatile analyte molecules for mass analysis using a photoabsorbing,low-sublimation temperature matrix. These matrix molecules provide ameans for desorbing and ionizing nonvolatile, nonthermally-labileorganic molecules such as biomolecules and synthetic polymers.Minimizing fragmentation of the parent analyte ion and/or reducingadduct formation leads to increased detection sensitivity and/orincreased resolution and/or extension of the usable mass range.

The deleterious effects associated with widening kinetic energy spreads,fragmentation and the formation of matrix-analyte adducts are reduced byemploying a matrix system, as disclosed herein, having lowerintermolecular binding energies associated with increased volatility.Lower binding energies can reduce fragmentation by minimizing theinternal energy of the desorbed analyte, and can reduce adduct formationby lowering the binding energy of the analyte with its surroundingmolecules. The desorption of a volatile matrix at room temperature butcooled to maintain low vapor pressure in the mass spectrometer may alsorequire less energy. Because a vacuum is required for the massspectrometry, volatile, crystalline matrices which sublimate orevaporate readily at room temperature are typically cooled to reducetheir vapor pressures to practical levels, which is below about 10⁻⁵Torr in the desorption plume. This consequently means that the analyteinternal energy may also be lower. It is therefore an advantage of thepresent invention to use liquids or low sublimation temperature solidsas matrices because such systems generally enable lowerdesorption/ionization temperatures.

The present invention relates to a method for volatilization and massspectrometric analysis of nonvolatile, or nonthermally labile, largeorganic molecules including biomolecules such as nucleic acids, forexample, DNA and RNA; proteins and peptide nucleic acids (PNA);oligosaccharides, and other high molecular weight polymers.

The invention generally provides a method for determining the mass of alarge organic molecule. The method typically includes contacting a largeorganic molecule, the mass of which one desires to determine, with aphotoabsorbing, or light absorbing, low-sublimation temperature matrixto produce a matrix:molecule mixture. This contacting step may becarried out by dissolving the large organic molecule to be analyzed in asolution containing the matrix. The matrix:molecule mixture is thenirradiated by a light source, such as a laser, to desorb, ionize, andproduce an ionized large organic molecule. The ionized large organicmolecule is then separated from other constituents, such as thematrix:molecule mixture or other matrix:molecule adducts, using massspectrometry and the mass of the ionized large organic moleculedetermined. While any mass spectrometry is contemplated for use with thepresent invention, time-of-flight mass spectrometry is preferred.

The matrix:molecule mixture typically comprises a physical mixture ofthe matrix with the molecule to be analyzed. It may or may not containadducts of the matrix with the molecule. Although if adducts are formed,they will typically be only weakly associated such that they may bereadily dissociated upon irradiation, desorption, and ionization.

As used herein the term “a” encompasses embodiments wherein it refers toa single element as well as embodiments including one or more of suchelements.

In performing the mass spectrometry, it is preferable to use a cooledsample stage. Generally, the sample stage is cooled to less than 273° K,typically to from about 150° K to 200° K or to about 180 K. While it iscontemplated that the sample stage may be cooled by any suitable means,it may typically be cryongenically cooled by liquid nitrogen.

In creating the matrix:molecule mixture, for example, by dissolving thelarge organic molecule in a solution containing the matrix, one of skillin the art will understand that the solution containing the matrix maygenerally contain one or more solvents. Preferably the solvents will bewater and/or organic solvents, such as ethanol, methanol, toluene,acetone, and acetonitrile. After the matrix:molecule mixture is formed,the solvents are substantially evaporated, typically to dryness. Inpreferred embodiments, the solvents are evaporated at room temperature.After evaporating the solvent, the resulting solid or crystallinemolecule-matrix mixture is cooled to a vapor pressure between about10⁻¹⁰ Torr and about 10⁻⁵ Torr.

The matrix for use in the present invention is generally a volatile,light-absorbing, hydroxy-bearing matrix. As used herein, volatilematrices are those that are volatile at room temperature at ambient orreduced pressures. In preferred aspects, the matrix may be a phenol, ahydroxyquinoline, or a hydroxynaphthalene. Where the matrix is a phenol,it will preferably be 4-nitrophenol. Where the matrix is ahydroxyquinoline, it will preferably be 8-hydroxyquinoline. It is alsogenerally preferred that the matrix have a molecular weight of betweenabout 90 Da and about 400 Da. Different classes of analyte molecules mayalso require different matrix systems. The matrix should typically notreact or interact strongly with the analyte and the analyte should besoluble in the matrix crystals.

In particular embodiments the matrix has a high sublimation rate betweenthe temperatures of 20° C. to 200° C. (or a low sublimationtemperature). The low-sublimation temperature matrix may typically havea sublimation rate at room temperature of at least 0.1 μm·min⁻¹ at apressure of about 10⁻⁵ Torr or less and preferably the sublimation rateat these conditions is from about 0.01 μm·min⁻¹ to about 0.1 mm·min⁻¹.Also provided are embodiments where the matrix is a crystalline solid.

As used herein the terms “photo absorbing” or “light absorbing” refer tothe ability of the matrix to absorb the desorption light sufficientlystrong to aid in the desorption and ionization of the large organicmolecule. Typically the matrices will absorb light between thewavelengths of approximately 200 nm and approximately 20,000 nm althoughit will be understood that this absorption is not continuous. It isfurther preferred that the photoabsorbing matrix have an absorptioncoefficient greater than about 10 L·cm⁻¹·mol⁻¹, up to and including anabsorption coefficient of 10⁶ L·cm⁻¹·mol⁻¹, at the wavelength of thedesorbing and ionizing radiation. The method of the invention is usefulfor determining the mass of virtually any large organic molecule. Forexample, the mass of a polymer may be determined using the methods ofthe invention. In preferred aspects of the invention, the polymer to beanalyzed will be a biopolymer, such as a nucleic acid, a polypeptide, apeptide nucleic acid (PNA), an oligosaccharide, or a mass-modifiedderivative thereof. Where the molecule to be analyzed is a nucleic acid,it will be understood that it may be, for example, a DNA or an RNA.

The analyte should typically be purified to minimize the presence ofsalt ions and other molecular contaminants. These impurities may reducethe intensity and quality of the mass spectrometric signal to a pointwhere either (i) the signal is undetectable or unreliable, or (ii) themass accuracy and/or resolution is below the value necessary for theparticular application, such as to detect the type of polymorphismexpected or sequence the analyte. A preferred method to purify theanalyte is to immobolize it on a solid support and wash it removeimpurities, such as sodium and potassium ions. The analyte may then bereleased from the solid support and contacted with the matrix.

The size of the analyte to be analyzed should also be within the rangewhere there is sufficient mass resolution and accuracy. Mass accuracyand resolution significantly degrade as the mass of the analyteincreases. Currently, the detection of single nucleotide polymorphisms(SNPs) above said mass value is difficult above a mass of approximately30,000 Da for oligonucleotides (˜100 bases) although this range mayincrease with further advances in MS-related technology. Third, becauseall molecules within a sample are visualized during mass spectrometricanalysis (i.e. it is not possible to selectively label and visualizecertain molecules and not others as one can with gel electrophoresismethods), samples may preferably be partitioned prior to analysis toremove unwanted products from the spectrum.

It is contemplated that the method of the invention will allow for themass determination of any large organic molecule having a mass ofgreater than about 1,000 Da. More specifically, one may determine themass of a molecule having a mass of greater than about 27,000 Da,greater than about 30,000 Da, greater than about 50,000 Da, greater thanabout 75,000 Da, greater than about 100,000 Da, greater than about150,000 Da, greater than about 175,000 Da, greater than about 200,000Da, greater than about 250,000 Da, or even greater than about 315,000Da. The organic molecule will typically have a mass of less than5,00,000 Da, 3,000,000 Da or 1,000,000 DA. In some embodiments, theorganic molecule may have a mass of less than 500,000 or 300,000Daltons.

To perform the desorbing step, one will generally expose thematrix:molecule mixture to a source of energy to desorb the largeorganic molecule from the matrix. The source of energy used fordesorption of the large organic molecule will preferably be a laserbeam. The laser beam used to desorb and ionize the large organicmolecule may be any laser but is preferably a pulsed laser. Typically,the desorption step will include applying an energy of about 20 kVfollowed by a pulse of energy of about 2.7 kV. Preferably, the pulse ofenergy comprises light having a wavelength of about 355 nm. The mass ofthe large organic molecule may then be determined by summing the massspectra over a number of laser pulses, preferably about 200 laser pulsesor about 1000 laser pulses, or any number of pulses therebetween, suchas, for example, about 250 laser pulses, about 300 laser pulses, about350 laser pulses, about 500 laser pulses, about 750 laser pulses, etc.Of course, it is contemplated that one may sum the mass spectra of lessthan about 200 pulses or more than about 1000 pulses, but it will beunderstood that lower numbers of pulses, especially very low numbers ofpulses such as 10 or 20 or 50 pulses, etc., may give less accurateresults, and higher numbers of pulses becomes unnecessarily repetitiveand lower the efficiency and cost-effectiveness of the method.

In another aspect, the invention also provides a method for preparing asample of large organic molecules for mass spectral analysis. Thismethod typically includes providing a solution comprising a largeorganic molecule to be analyzed, a matrix molecule comprising avolatile, light-absorbing hydroxy-bearing matrix molecule, and asolvent, and evaporating the solvent to provide a solid crystallinematrix containing the molecule to be analyzed.

The present invention applies to MALDI mass spectrometry of all classesof nonvolatile, large organic compounds, with synthetic polymers andbiopolymers preferred. The present invention is particularly preferredfor mass analysis of biopolymers such as nucleic acids, proteins, PNAsand oligosaccharides due to the fragile nature of these molecules. Themethod utilizes pulsed laser desorption/ionization mediated by a matrixfollowed by mass spectrometric separation and detection of the analytemolecules. The matrix may be a crystalline solid or a liquid at roomtemperature, with crystalline solids being preferred. The preferredmatrix has a high sublimation rate in vacuum at room temperature andabsorbs the desorption light strongly.

Therefore in accordance with the present invention, there is providedcrystalline solid, light absorbing compounds having hydroxyfunctionalities, but not carboxylic functionalities, for use as a matrixin mass analysis. In preferred embodiments the matrix compounds may bephenols, hydroxyquinolines or hydroxynaphthalenes. The crystallinesolids, 8-hydroxyquinoline and 4-nitrophenol, which are volatile at roomtemperature, are particularly preferred as matrices in accordance withthe present invention.

The less energetic, more facile desorption/ionization from thesevolatile matrices minimizes fragmentation and extends the high masslimit for generation of intact analyte molecules. These crystallinematrices exhibit increased sensitivity for detection of both low(8-hydroxyquinoline) and high (4-nitrophenol) molecular weight analytes.Analyte molecules, including DNA, exceeding 250 kDa molecular weight canbe detected by this method.

There is provided embodiments where the analyte is a large organicmolecule of greater than about 1,000 Da. Also provided are embodimentswhere the large organic analyte is a polymer. In certain embodiments thepolymer is a biopolymer. In further embodiments the biopolymer is apolynucleic acid, and in still further embodiments the biopolymer is anoligonucleotide. Additionally provided are embodiments where thebiopolymer is a protein, polypeptide, or oligosaccharide.

In yet other embodiments, the sample is placed on a cooled sample stagein order to maintain a low vapor pressure of the sample in the vacuumchamber of the mass spectrometer. The sample stage is cooled below about273° K, more typically between about 170 to about 190° K, and mosttypically to about 180° K.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 is a laser desorption/ionization time-of-flight mass spectrum ofa mixture of single-stranded DNA oligomers 89, 90, and 91 nucleotides inlength obtained using 8-hydroxyquinoline as the matrix. The laserwavelength was 355 nm.

FIG. 2 is a laser desorption/ionization time-of-flight mass spectrum ofa double-stranded PCR product at 315 kDa per strand (greater thanapproximately 1000 nucleotides in length) using a 4-nitrophenol matrix.The laser wavelength was 355 nm.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In accordance with the present invention, methods are provided for thepreparation of samples for analysis by mass spectroscopy to minimizeundesired fragmentation. Select light absorbing molecules, containinghydroxy functionalities (but not carboxylic functionalities) and havingsignificant sublimation rates at room temperature under vacuum, are usedas matrices in MALDI mass spectrometry. Hydroxy functionalities offeradvantage over carboxylic functionalities due to their increased acidityin the excited state (Huppert et al., 1981) and also typically providelower intermolecular binding energies to increase volatility.Representative examples of matrix compounds include, but are not limitedto, hydroxyquinolines, phenols, and hydroxynaphthalenes.

Samples are prepared by dissolving the analyte in a solution containingthe matrix molecule, with the bulk of the solution being one or moresolvents which are subsequently allowed to evaporate before massanalysis begins. Typically, the analyte will be present in the solutionat a concentration of about 0.05 M to about 1.0 M.

The solvent evaporation may be conducted at a temperature range of about20° C. to about 30° C., with room temperature, about 25° C. being mostpreferred. The evaporation results in the formation of a crystallinematrix, composed in part (between about 30% by weight to about 100% byweight) of the subject matrix molecule. Typically the matrix molecularweight is greater than about 90 Da, preferably between about 90 Da andabout 400 Da. Due to the high volatility of these matrix molecules undervacuum at room temperature, the samples must be cooled in the massspectrometers vacuum system to a vapor pressure between about 10⁻¹⁰ Torrand about 10⁻⁵ Torr, but not exceeding about 10⁻⁵ Torr. These matrixmolecules are termed herein as volatile, light-absorbing,hydroxy-bearing matrix molecules. As used herein the term volatilerefers to a molecule having a sublimation rate at room temperature ofgreater than or equal to 0.1 μm·min⁻¹ at a pressure of about 10⁻⁵ Torror less, and the term light absorbing refers to a molecule having anabsorption coefficient greater than about 10 l·cm⁻¹·mol⁻¹.

Two low-sublimation-temperature molecules in particular functioneffectively as matrices for MALDI of nonvolatile organic molecules fordetection by mass spectrometry. The compounds, 8-hydroxyquinoline (8HQ)and 4-nitrophenol (4NP), both contain a hydroxy functional group. Theformer is especially effective for high-resolution analysis of DNA lessthan approximately 100 nucleotides (30 kDa), and the latter isespecially effective for sensitive detection of higher mass molecules.

Compounds contemplated for analysis using the present invention includea vast array of large organic molecules. As used herein, the term “largeorganic molecule” refers to a compound having a molecular weight ofgreater than about 1000 Da. Also as used herein, the term “nonvolatile”refers to a molecule which, when present in its pure, neat form andheated, does not sublimate intact to any significant extent. Alsoincluded in the definition of nonvolatile compounds are compounds which,when present in their pure neat form, cannot be practically analyzed bymass spectrometry when conventional gas chromatography methods areemployed in the sampling process. Representative of such organiccompounds are polynucleic acids, polypeptides, oligosaccharides, PNAsand synthetic polymers. Polymeric compounds are also contemplated foranalysis using the present invention. In particular biopolymers whichare subject to fragmentation during mass analysis. Representativebiopolymers include polymers of amino acids, nucleic acids, saccharides,carbohydrates and polypeptides.

The mass spectrometry may be accomplished by one of several techniquessuch as time-of-flight, magnetic sector or ion trap. Preferably, themass spectrometry technique for use with the present invention will betime-of-flight.

The volatility of the matrix crystals necessitates that the sample stageof the mass spectrometer be cooled to substantially below roomtemperature where the sublimation rate is between about 0.1 μm·min⁻¹ andabout 0.1 mm·min⁻¹. A preferred approach is to use a liquid-nitrogencooled sample stage, accomplished by flowing liquid nitrogen through acopper sample holder. Thus, the sample is cooled to less than 273° K,preferably between about 170 and 190° K or to about 180° K.

Wavelengths from the ultraviolet to infrared may be employed, dependingon the cooled matrix being analyzed. Generally, one of skill in the artwill understand that the appropriate wavelength will be one where lightabsorption is significant for the molecule being analyzed.

The disclosed low-sublimation temperature matrices and methods for usingthem to determine the mass of a large organic molecule or prepare alarge organic molecule for mass spectral analysis may be used in avariety of MS applications, such as MS sequencing of nucleic acids; MSanalysis of single nucleotide polymorphisms (SNPs); and MS analysis ofsimple sequence repeats (SSRs), short tandem repeats (STRs), andmicrosatellite repeats (MRs).

For example, the methods disclosed herein may be used in nucleic acidsequencing methods involving obtaining nucleic acid fragments using afour base Sanger sequencing reaction, performing MS on the products anddetermining the nucleic acid sequence from the mass differences betweenthe peaks. The nucleic acid fragments may be obtained by hybridizing aDNA primer to a DNA template and extending the primer by a DNApolymerase in the presence of deoxy- and dideoxy-nucleotides. The DNAtemplate may generally contain the DNA fragment to be sequenced and aregion complementary to the primer. The DNA primers may also contain abiotin which allows for capture to a solid phase and a single,chemically cleavable internal linkage (such as a 5′-or3′-(S)-phosphorothioate linkage which is cleavable by a silver ioncatalyzed reaction). The cleavage chemistry of the internal linkagecombined with the biotin capture are described in U.S. Pat. No.5,700,642, incorporate herein by reference.

The nucleic acid fragments may be further processed prior to MSanalysis. Generally, these processing steps involve binding the nucleicacid fragments to a streptavidin solid support, washing the boundfragments, and cleaving at the internal cleavage site to release thenucleic acid fragment from the solid support. Typically the boundfragments are first washed with a denaturant, such as aqueous NaOH, toremove unbound DNA and enzyme and then with a series of ammonium acetatewashes. Following cleavage, the cleaved extension products may beprepared for MS analysis by drying; mixing the solid residue with thematrix material and ammonium citrate solution; spotting the mixture bypipette onto a plate; and allowing the mixture to dry.

The methods for MS SNP analysis are very similar to the DNA sequencingmethods except that only dideoxynucleotides are employed.

These low-sublimation temperature matrices may also be used foranalyzing SSRs, STRs, and MRs involving the determination of the numberof repetitive units contained in amplification products by MS. Theamplification products are typically obtained by hybridizing a DNAprimer to a DNA target molecule and extending the primer by a DNApolymerase. Similar to the sequencing methods, the DNA primer contains aregion complementary to the DNA target molecule adjacent to the SSR-,STR-, or MR-containing region. The primer may also contain biotin andinternal cleavable linkages.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials and Methods

A time-of-flight mass spectrometer similar to that previously describedby Wu et al. (1994) and Hunter et al. (1997) was used, having pulseddelayed ion extraction. The sample stage was floated at 20 kV, and aftersome delay time (approximately several hundred nanoseconds, dependent onmass), ions were extracted by a 2.7 kV pulse and focused into a 1-meterflight tube. The signal output from the dual microchannel plate detectorwas amplified and digitized with 5 ns time resolution.

Laser wavelengths of either 355 or 266 nm were employed fordesorption/ionization in the examples below. Comparable positive andnegative ion signals were observed from oligonucleotide analytes.

The temperature of the sample on a liquid nitrogen-cooled sample stagewas maintained at approximately 180 K as measured by thermocouple wires,low enough to maintain a matrix vapor pressure of less than 10⁻⁵ Torr.

Example 2 DNA Oligomer Analysis Employing 8-Hydroxyquinoline as a Matrix

The preparative solution for the 8HQ matrix began by using 0.2 M 8HQ in1:1 (volume) acetone:butanone. To reduce alkali-metal adduct ionformation, to that initial 8HQ solution was added an equal volume of 50mM aqueous diammonium citrate, resulting in a 25 mM final diammoniumcitrate concentration and 0.1 M 8HQ concentration. 8HQ is known tochelate trace amounts of metal ions, especially copper, but the additionof CDTA (trans-1,2-diaminocyclohexane-N,N,N′,N′ tetraacetic acidmonohydrate) effectively suppressed copper adducts in the mass spectrum;a small aliquot of concentrated CDTA was added to a much larger volumeof the 8HQ solution to yield a 10 mM CDTA concentration.

The oligonucleotide sample was obtained from polymerase chain reaction(PCR) amplification of a short tandem repeat sequence at the human TH01(tyrosine hydroxylase gene) locus. One of the strands was captured,denatured, washed, then released to produce single-stranded products. Analiquot of aqueous solution of this TH01 oligonucleotide (estimated 10pmol quantity) was first evaporated in a vacuum evaporator to remove thewater, and then one microliter of the matrix solution was added to thedried DNA. This resulting solution was pipetted onto a silicon substratemounted on a copper sample holder. After air-drying of the solvent andresultant crystallization of the matrix, the sample was placed on thecryogenically-cooled sample stage in the mass spectrometer.

8HQ is an effective matrix for high resolution studies of DNA oligomersless than approximately 100 nucleotides in length. FIG. 1 illustratesthe mass resolution attainable for single-stranded oligonucleotides ofabout 27 kDa using 355 nm pulsed laser light for desorption and summingmass spectra over 200 laser pulses. DNA oligomers containing 89, 90, and91 nucleotides have a mass resolution (m/Δm) of 650, 625, and 700,respectively at full width at half height. Spectra of oligonucleotidesin 8HQ matrix typically have a low background ion signal and highsignal-to-noise levels.

Example 3 DNA Oligomer Analysis Employing 4-Nitrophenol as a Matrix

The preparative solution for the 4NP matrix was 0.5 M 4NP in 1:1(volume) methanol:water containing diammonium citrate at 50 mM finalconcentration. One microliter of the matrix solution was added to driedDNA which was a double-stranded PCR product estimated at 10 pmolquantity derived from an unknown cDNA insert in a vector. This resultingsolution was pipetted onto a silicon substrate mounted on a coppersample holder. After air-drying of the solvent and resultantcrystallization of the matrix, the sample was placed on thecryogenically-cooled sample stage in the mass spectrometer. FIG. 2 isthe resulting time-of-flight mass spectrum using 355 nm laser light fordesorption and summing over 1000 laser pulses yielding an estimated massof 315 kDa which corresponds to an estimated number of bases exceeding1,000. The width of the peak originates in part from the mass differenceof the two complementary DNA strands (denatured during analysis) andpartly from adduct formation as well as fragmentation. DNA oligomershave not previously been reported to be detected in this size range.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

U.S. Pat. No. 4,920,264 to Becker.

U.S. Pat. No. 5,118,937 to Hillenkamp et al.

U.S. Pat. No. 5,135,870 to Williams et al.

Abrams et al., “Comprehensive Detection of Single Base Changes in HumanGenomic DNA Using Denaturing Gradient Gel Electrophoresis and a GCClamp,” Genomics, 7:463-475, (1990).

Gimon et al., “Are Proton Transfer Reactions of Excited States Involvedin UV Laser Desorption Ionization?,” Organic Mass Spectrometry,27:827-830 (1992).

Hunter et al., “Cryogenic Frozen Solution Matrices for Analysis of DNAby Time-of-Flight Mass Spectrometry,” Analytical Chemistry, 69:3608-12(1997).

Huppert et al., “Laser Studies of Proton Transfer,” Advances in ChemicalPhysics, 47:643-679 (1981).

Liu et al. “Use of a Nitrocellulose Film Substrate in Matrix-AssistedLaser Desorption/Ionization Mass Spectrometry for DNA Mapping andScreening,” Analytical Chemistry, 67:3482-3490 (1995).

Nelson et al., “Detection of Human IgM at m/z˜1 MDa,” RapidCommunications in Mass Spectrometry, 9:625 (1995).

Orita et al., “Detection of Polymorphisms of Human DNA by GelElectrophoresis as Single-Strand Conformation Polymorphisms,” Proc.Natl. Acad. Sci. USA, 86:2766-2770, 1989

Saleeba et al., “Chemical Cleavage of Mismatch to Detect Mutations,”Methods Enzymology, 217:286-295 (1993).

Spengler et aL, “Laser Mass Analysis in Biology,” Ber. Bunsenqes Phys.Chem., 93(3):396-402, (1989).

Tanaka et al., “Protein and Polymer Analyses up to m/z 100 000 by LaserIonization Time-of-flight Mass Spectrometry,” Rapid Commun. in MassSpectrometry, 2:151-153 (1988).

Tang et al. “Detection of 500-Nucleotide DNA by Laser Desorption MassSpectrometry,” Rapid Communications in Mass Spectrometry, 8:727-730(1994).

Wu et al. “Matrix-Assisted Laser Desorption Time-of-Flight MassSpectrometry of Oligonucleotides Using 3-Hydroxypicolinic Acid as anUltraviolet-Sensitive Matrix,” Rapid Communications in MassSpectrometry, 7:142-146 (1993).

Wu et al. “Time-of-Flight Mass Spectrometry of UnderivatizedSingle-Stranded DNA Oligomers by Matrix-Assisted Laser Desorption,”Analytical Chemistry, 66:1637-1645 (1994).

Youil et al., “Screening for Mutations by Enzyme Mismatch Cleavage withT4 Endonuclease VII,” Proc. Natl. Acad. Sci. USA, 92:87-91 (1995).

What is claimed is:
 1. Components for the preparation of a large organicmolecule for analysis by mass spectrometry, the components comprising: avolatile matrix comprising a photoabsorbing low-sublimation temperaturematrix; one or more solvents; and a sample stage capable of beingcooled.
 2. The components of claim 1, wherein the volatile matrixcomprises a volatile, light-absorbing, hydroxy-bearing matrix.
 3. Thecomponents of claim 1, wherein the volatile matrix is selected from thegroup consisting of phenols, hydroxyquinolines, and hydroxynaphthalenes.4. The components of claim 3, wherein the volatile matrix comprises4-nitrophenol.
 5. The components of claim 3, wherein the volatile matrixcomprises 8-hydroxyquinoline.
 6. The components of claim 1, wherein thevolatile matrix has a molecular weight of between about 90 Da and about400 Da.
 7. The components of claim 1, wherein a sublimation rate of thevolatile matrix is at least 0.1 μM/min at a pressure of about 10⁻⁵ Torr.8. The components of claim 1, wherein a sublimation rate of the volatilematrix is between about 0.01 μM/min to about 0.1 μM/min.
 9. Thecomponents of claim 1, wherein the volatile matrix comprises aphotoabsorbing matrix capable of absorbing light having a wavelengthbetween about 200 nm and 20,000 nM.
 10. The components of claim 1,wherein the volatile matrix comprises a photoabsorbing matrix having anabsorption coefficient between about 10 L/cm/mol and about 10⁶ L/cm/mol.11. The components of claim 1, wherein the one or more solvents compriseone or more of water, methanol, ethanol, toluene, acetone, oracetonitrile.
 12. The components of claim 1, wherein the one or moresolvents comprises diammonium citrate,trans-1,2-diaminocyclohexane-N,N,N′,N′ tetraacetic acid monohydrate, ora combination thereof.
 13. The components of claim 1, wherein thesolvent comprises 1:1 acetone:butanone.
 14. The components of claim 1,wherein the sample stage comprises a liquid nitrogen-cooled samplestage.
 15. The components of claim 1, wherein the sample stage comprisesa copper sample holder.
 16. The components of claim 1, wherein thesample stage comprises a silicon substrate.
 17. The components of claim1, wherein the large organic molecule comprises one or more biomoleculesor synthetic polymers.
 18. The components of claim 17, wherein the oneor more biomolecules comprise a nucleic acid, a polypeptide, a peptidenucleic acid, an oligosaccharide, or a mass-modified derivative thereof.19. The components of claim 18, wherein the nucleic acid comprises DNAor RNA.
 20. The components of claim 1, further comprising: a solidsupport capable of binding the large organic molecule; and a washingsolvent.
 21. The components of claim 20, wherein the large organicmolecule comprises biotin and the solid support comprises abiotin-binding moiety.
 22. The components of claim 21, wherein thebiotin-binding moiety comprises streptavidin.
 23. The components ofclaim 1, further comprising one or more of a DNA primer, a biotin-linkedDNA primer, a DNA polymerase, a denaturant, aqueous NaOH, ammoniumacetate, and ammonium citrate.